Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. An additional example of modulation of RNA target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
The synthesis of 5′-substituted DNA and RNA derivatives and their incorporation into oligomeric compounds has been reported in the literature (Saha et al., J. Org. Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020). The 5′-substituted monomers have also been made as the monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).
A genus of modified nucleosides including optional modification at a plurality of positions including the 5′-position and the 2′-position of the sugar ring and oligomeric compounds incorporating these modified nucleosides therein has been reported (see International Application Number: PCT/US94/02993, Published on Oct. 13, 1994 as WO 94/22890).
The synthesis of 5′-CH2 substituted 2′-O-protected nucleosides and their incorporation into oligomers has been previously reported (see Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924).
Amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to 3′) comprises a 2′-OCH3 and a 5′-(S)—CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290).
A genus of 2′-substituted 5′-CH2 (or O) modified nucleosides and a discussion of incorporating them into oligonucleotides has been previously reported (see International Application Number: PCT/US92/01020, published on Feb. 7, 1992 as WO 92/13869).
The synthesis of modified 5′-methylene phosphonate monomers having 2′-substitution and their use to make modified antiviral dimers has been previously reported (see U.S. patent application Ser. No. 10/418,662, published on Apr. 6, 2006 as US 2006/0074035).
The synthesis of 5′-methylenebis(phosphonate) monomers having a 2′-OCH3 (or OH) and their use to make dinucleotide mRNA cap analogs have been previously described (see Rydizk et al., Org. Biomol. Chem., 2009, 7, 4763-4776 and Myers et al., J. Org. Chem., 1965, 30, 1517-1520). Other nucleosides comprising 5′-methylene or 5′-difluoromethylene bisphosphonates have also been reported wherein the hydroxyl groups at the 2′ or/and 3′ positions are protected or substituted (see published International Applications WO 94/29331, WO 98/15563 and WO 2006/038865).
A genus of modified 5′-diphosphate and 5′-triphosphate nucleosides and their potential use as antiviral agents has been described (see published International Application WO 03/072757). Other modified 5′-diphosphate mononucleosides and their use to make dimeric compounds have been previously reported (see published International Application WO 2007/020018 and U.S. Pat. No. 7,018,985, issued on Mar. 28, 2006; U.S. Pat. No. 7,101,860, issued on Sep. 5, 2006 and U.S. Pat. No. 7,132,408, issued on Nov. 7, 2006).
The synthesis of 5′(S)- and 5′(R)-methyl (or propyl) ribonucleosides having a 5′-pyro-phosphate group (P—O—P) have been described along with their physico-chemical and biological property studies (see Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Hai et al., J. Med. Chem., 1982, 25, 1184-1188; David et al., Journal of Chemical Society Perkin Trans I: Org. Bioorg. Chem., (1972-1999) (1982), 2, 385-393 and Mikhailov et al., Nucleosides Nucleotides, 1991, 10, 339-343).
Various 5′-monophosphate and 5′-triphosphate nucleosides having a 5′-acylaminomethyl group (5′-CH2NH(C═O)CH3) (and analogs thereof) have been reported in the literature and were further evaluated for biological activities (see Hampton et al., J. Med. Chem., 1979, 22, 621-631 and J. Med. Chem., 1976, 19, 1371-1377; Kappler et al., J. Org. Chem., 1975, 40, 1378-1385, J. Med. Chem., 1987, 30, 1599-1603 and J. Med. Chem., 1990, 33, 2545-2551).
The synthesis of 5′-monophosphate and 5′-triphosphate DNA and RNA monomers, oligomers comprising these monomers, Tm evaluations of these oligomers and their preparation as fluorescent probes have been described (see PCT International Application WO 00/14101, published on Mar. 16, 2000).
The preparation of deoxyribonucleosides and ribonucleosides comprising a 5′-imidodiphos-phate group (P—NH—P) and their biological evaluations have been previously reported (see Tomasz et al., Nucleic Acids Research, 1988, 16, 8645-8664).